Kokumi-imparting agent

ABSTRACT

The present invention encompasses a method for screening for a kokumi-imparting substance by using the calcium receptor activity as an index, a composition containing a kokumi-imparting substance obtained by the screening method, a method for producing food or drink imparted with kokumi, and food or drink imparted with kokumi.

This application is a divisional under 35 U.S.C. §120 of U.S. patentapplication Ser. No. 12/117,027, filed May 8, 2008, which claimspriority under 35 U.S.C. §119 to Japanese Patent Application No.2005-325300, filed on Nov. 9, 2005, U.S. Provisional Patent ApplicationNo. 60/738,562, filed on Nov. 22, 2005, Japanese Patent Application No.2006-188458, filed on Jul. 7, 2006, and U.S. Provisional PatentApplication No. 60/807,831, filed on Jul. 20, 2006, and is acontinuation application under 35 U.S.C. §120 to PCT Patent ApplicationNo. PCT/JP2006/322694, filed on Nov. 8, 2006, the contents of which areincorporated by reference in their entireties. The Sequence Listingfiled electronically herewith is also hereby incorporated by referencein its entirety (File Name: 2010-11-23T_US-259D_Seq_List; File Size: 1KB; Date Created: Dec. 7, 2010).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of screening for a substancewhich imparts kokumi, a composition containing a substance which impartskokumi obtained by the screening method, a method for producing a food,seasoning, or drink imparted with kokumi, and a food or drink impartedwith kokumi.

2. Brief Description of the Related Art

The calcium receptor (also called the calcium sensing receptor, CaSR)contains 1078 amino acids, and is classified as in class C of theseven-transmembrane receptors (G protein-coupled receptor; GPCR). Thecloning of the gene for the calcium receptor was reported in 1993(Nature, 1993 Dec. 9; 366(6455):575-80). The calcium receptor is knownto cause various cellular responses through elevation of theintracellular calcium levels, etc., when activated with calcium, etc.The sequence of the human calcium receptor gene is registered withGenBank (Accession No. NM_000388), and is well conserved among manyanimal species.

The calcium receptor may promote or suppress various biologicalfunctions. Therefore, therapeutic agents which act as activators orinhibitors of the calcium receptor are appropriately used in thetreatment of neurological diseases, hepatic diseases, cardiovasculardiseases, digestive system diseases, and other diseases, depending onthe pathological conditions. For example, the calcium receptor is ableto detect increased levels of blood calcium in the parathyroid, andsuppress secretion of the parathyroid hormone (PTH) to correct the bloodcalcium level. Therefore, reduction of the blood calcium level is anexpected effect of administration of a calcium receptor activator. Ithas been reported that when a calcium receptor activator is used totreat secondary hyperparathyroidism in a hemodialysis patient, the PTHlevel is reduced without the calcium and phosphorus levels increasing.

Since functional studies of the calcium receptor have been conductedprimarily during calcium homeostasis, applications so far typicallyconcern bone metabolic diseases in which calcium regulation is involved.However, through analysis of genetic expression, it is now known thatthe calcium receptor is widely distributed in living bodies in additionto the parathyroid and kidney tissues (J. Endocrinol., 2000 May,165(2):173-7 and Eur. J. Pharmacol., 2002 Jul. 5, 447(2-3):271-8), andthe possibility that the calcium receptor is involved in many variousbiological functions and the etiology of many diseases has beenproposed. For example, the calcium receptor is thought to be involved inthe function of the liver, heart, lung, alimentary canal, lymphocyte,and pancreas. It has been confirmed that the calcium receptor isexpressed in a wide range of tissues by analyses based on RT-PCR usingRNAs extracted from rat tissues. Therefore, the increased importance ofactivators and inhibitors of the calcium receptor in variousapplications is becoming recognized.

Moreover, cations such as gadolinium, basic peptides such aspolyarginine, polyamines such as spermine, amino acids such asphenylalanine, and so forth have been reported to be calcium receptoractivators (Cell Calcium., 2004 March, 35(3):209-16).

Although many specific calcium receptor activators have been developedas described above, few of these compounds are native to living bodies,and those that are native have very low activities. Therefore,therapeutic agents containing these activators pose serious problemsincluding side effects, permeability, and sufficient activity. Forexample, although it is known that amino acids act on calcium receptors,their use as calcium receptor activators is difficult due their veryweak activity. Moreover, although macromolecules such as polyargininehave been reported to be an activator as described above, the activatorfunction is based on their actions as polyvalent cations, which haveirregular structures. That is, peptides having a specific structure arenot known to be useful as a calcium receptor activator.

In the field of foodstuffs, substances having specific tastes have beenused for many years. In particular, substances having the five basictastes, namely, sweet, salty, sour, bitter, and umami (a delicioustaste) have been widely used as seasonings. Substances which enhancethese basic tastes have also been widely used. One taste that does notfall within these five basic tastes is “kokumi”. Kokumi means a tastethat is not one of the five basic tastes. Kokumi is a taste that notonly enhances the five basic tastes but also enhances the marginaltastes of the basic tastes, such as thickness, growth (mouthfulness),continuity, and harmony. Several methods for imparting kokumi have beenreported so far. Substances that have been reported to impart kokumiinclude glutathione (Japanese Patent No. 1464928), heated products ofgelatin and tropomyosin (Japanese Patent Laid-open Publication (KOKAI)No. 10-276709), sulfone group-containing compounds (Japanese PatentLaid-open Publication (KOKAI) No. 8-289760), a peptide containing theAsn-His sequence (WO2004/096836), and so forth.

Although the development of various kokumi-imparting substances has beenattempted as described above, and those that have been commercializedhave been mainly extracts of natural products, there are presently veryfew examples of isolation of a pure kokumi component from an extract ofnatural product, such as glutathione andN-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine.

Therefore, the development of highly effective, safe, and inexpensivekokumi-imparting substances is desired, and a convenient and highlysensitive method of screening for a kokumi-imparting substance is neededfor that purpose.

SUMMARY OF THE INVENTION

An aspect of the present invention is to provide a convenient and highlysensitive method for screening for a kokumi-imparting substance, ahighly effective, safe and inexpensive kokumi-imparting agent,compositions containing these substances or agents, a method forproducing food, seasoning, or drink imparted with kokumi, and food,seasoning, or drink imparted with kokumi.

It has been found that low molecular weight peptides, includingglutathione, are able to activate the calcium receptor. Moreover, sinceglutathione is known to be a kokumi-imparting substance, other lowmolecular weight peptides which are known to be activators of thecalcium receptor were evaluated for their ability to impart kokumi, andit was found that the low molecular weight peptides imparted kokumi. Thepresent invention was accomplished on the basis of these findings.

It is an aspect of the present invention to provide a method forscreening for a kokumi-imparting substance comprising utilizing calciumreceptor activity as an index.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the kokumi-imparting substance enhances a tasteselected from the group consisting of salty, umami, sweet, and sour.

It is a further aspect of the present invention to provide the method asdescribed above comprising a) reacting a calcium receptor and a testsubstance, b) detecting calcium receptor activity of the test substance,and c) measuring the kokumi-imparting effect of the test substancehaving calcium receptor activity.

It is a further aspect of the present invention to provide a compositioncomprising the kokumi-imparting substance obtained by the method asdescribed above.

It is a further aspect of the present invention to provide thecomposition as described above comprising a substance selected from thegroup consisting of γ-Glu-X-Gly, wherein X is an amino acid or an aminoacid derivative except for Cys, γ-Glu-Val-Y, wherein Y is an amino acidor an amino acid derivative, γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met,γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys,Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH₂,γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu,γ-Glu-Ile, γ-Glu-t-Leu, γ-Glu-Cys(S-Me), and combinations thereof.

It is a further aspect of the present invention to provide thecomposition as described above, wherein X is selected from the groupconsisting of Cys(SNO), Cys(S-allyl), Gly, Cys(S-Me), Abu, and Ser, andY is selected from the group consisting of Gly, Val, Glu, Lys, Phe, Ser,Pro, Arg, Asp, Met, Thr, His, Orn, Asn, Cys, and Gln.

It is a further aspect of the present invention to provide thecomposition as described above, which has the property of enhancing ataste selected from the group consisting of salty, umami, sweet, andsour.

It is a further aspect of the present invention to provide a foodcomposition comprising the composition as described above, and acomposition comprising at least one compound having calcium receptoractivation activity.

It is a further aspect of the present invention to provide the foodcomposition as described above, wherein the compound having calciumreceptor activation activity is selected from the group consisting ofcalcium, protamine, polyarginine, spermine, polylysine, glutathione,cinacalcet, and combinations thereof.

It is a further aspect of the present invention to provide a method forproducing a food or drink imparted with kokumi comprising adding one ormore of the compositions as described above to a food or drink to aconcentration of 1 mass ppb to 99.9 mass %.

It is a further aspect of the present invention to provide a method forproducing food or drink imparted with kokumi comprising adding aseasoning comprising the composition as described above at aconcentration of 1 mass ppb to 99.9 mass % to food or drink to aconcentration of 0.01 to 10 mass %.

It is a further aspect of the present invention to provide a food ordrink imparted with kokumi obtained by the method as described above.

It is a further aspect of the present invention to provide a compositioncontaining 1 mass ppb to 99.9 mass % of γ-Glu-Val-Gly and 1 mass ppb to99.9 mass % of a substance selected from the group consisting ofcalcium, protamine, polyarginine, spermine, polylysine, glutathione, andcinacalcet.

It is a further aspect of the present invention to provide a compositioncomprising 1 mass ppb to 99.9 mass % of a substance selected from thegroup consisting of glutathione, protamine, polylysine, GABA, salt formsthereof, and combinations thereof, and 1 mass ppb to 99.9 mass % ofcalcium or a salt form thereof.

It is a further aspect of the present invention to provide a compoundselected from the group consisting of:

a) γ-Glu-X-Gly, wherein X is selected from the group consisting of Asn,Gln, His, Lys, Orn, and Arg, and

b) γ-Glu-Val-Y, wherein Y is selected from the group consisting of Leu,Ile, Ser, Thr, Met, Cys, Asp, Asn, Gln, Lys, Orn, Arg, Phe, Tyr, Pro,Hyp, Trp, His, and Abu.

It is a further aspect of the present invention to provide a method forimparting kokumi to a food or drink comprising adding a compositionobtained by the method as described above to the food or drink.

It is a further aspect of the present invention to provide a method ofimparting kokumi to a composition comprising adding the kokumi-impartingsubstance obtained by the method described above to a composition.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the kokumi-imparing substance is selected fromthe group consisting of γ-Glu-X-Gly, wherein X is an amino acid or anamino acid derivative except for Cys, γ-Glu-Val-Y, wherein Y is an aminoacid or amino acid derivative, γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys,γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met,Glu-Cys, Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val,γ-Glu-Val-NH₂, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O),γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, γ-Glu-Cys(S-Me), and combinationsthereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein X is selected from the group consisting ofCys(SNO), Cys(S-allyl), Gly, Cys(S-Me), Abu, or Ser, and Y is selectedfrom the group consisting of Gly, Val, Glu, Lys, Phe, Ser, Pro, Arg,Asp, Met, Thr, His, Orn, Asn, Cys, and Gln.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the kokumi-imparing substance is γ-Glu-Val-Gly.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the composition is a food or drink.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the action of calcium on the calcium receptor byintroducing the human calcium receptor cRNA into Xenopus laevis oocytesby microinjection. The intracellular response currents were recordedwhen a calcium chloride solution was added at an arbitraryconcentration. The maximum intracellular currents were considered to bethe response current values. No response was observed in control oocytesmicroinjected only with distilled water.

FIG. 2 shows the action of L-amino acids on the calcium receptor byintroducing the human calcium receptor cRNA into Xenopus laevis oocytesby microinjection. The intracellular response currents were recordedwhen a 10 mM L-amino acid solution was added. The maximum intracellularcurrents were considered to be the response current values. No responsewas observed in control oocytes microinjected with only distilled water.

FIG. 3 shows the action of D-amino acids on the calcium receptor byintroducing the human calcium receptor cRNA into Xenopus laevis oocytesby microinjection. The intracellular response currents were recordedwhen a 10 mM D-amino acid solution was added. The maximum intracellularcurrents were considered to be the response current values. No responsewas observed in control oocytes microinjected with only distilled water.

FIG. 4 shows the action of peptides on the calcium receptor byintroducing the human calcium receptor cRNA into Xenopus laevis oocytesby microinjection. The intracellular response currents were recordedwhen a peptide solution was added at an arbitrary concentration. Themaximum intracellular currents were considered to be the responsecurrent values. No response was observed in control oocytesmicroinjected with only distilled water.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided is a convenient and highly sensitive method of screening for akokumi-imparting substance, a highly effective, safe, and inexpensivecomposition containing the kokumi-imparting agent obtained by thescreening method, a method for producing food, seasoning, or drinkimparted with kokumi, and food, seasoning, or drink imparted withkokumi.

The calcium receptor can also be referred to as the calcium sensingreceptor (CaSR), and belongs to class C of the seven-transmembranereceptors. The “calcium receptor activity” is when binding of asubstrate to the calcium receptor activates the guanine nucleotidebinding protein and, as a result, transmits one or more signals.Furthermore, the “calcium receptor activator” is a substance that actson the calcium receptor to activate the calcium receptor and, as aresult, controls the functions of cells expressing the calcium receptor.

“Kokumi” means a taste that is not one of the five basic tastes: sweet,salty, sour, bitter, and umami. Kokumi also means when a substancefunctions to enhance other properties of the five basic tastes, such asthickness, growth (mouthfulness), continuity, and harmony. Furthermore,a “kokumi-imparting agent” or “kokumi-imparting substance” refers to anagent or substance that can enhance one or more of the five basictastes, and also enhance other properties of the basic tastes, such asthickness, growth (mouthfulness), continuity, and harmony whichaccompany the basic tastes. Therefore, the kokumi-imparting agent canalso be used as a sweet taste-enhancing agent, salty taste-enhancingagent, sour taste-enhancing agent, bitter taste-enhancing agent, orumami-enhancing agent. As for the intensity of kokumi, the “first andmiddle taste” means the taste that is experienced from 0 to 4 secondsafter eating, and the “aftertaste” means the taste that is experienced 5seconds after eating.

All the amino acids and amino acid residues which make up the peptidesare L-isomers, unless otherwise specified.

Method for Screening for Kokumi-Imparting Substance

The method for screening for a kokumi-imparting substance ischaracterized by using calcium receptor activity as an index.Specifically, this screening method includes steps of reacting a calciumreceptor and a test substance, detecting calcium receptor activity ofthe test substance, and measuring the kokumi-imparting effect of thetest substances having calcium receptor activation activity.

The specific process steps of the screening method are exemplifiedbelow. However, the steps of the screening method are not limited tothese steps. The steps are as follows:

1) A test substance is added to a system which is set up to measure thecalcium receptor activity of the test substance, and the calciumreceptor activity is measured.

2) The calcium receptor activity of the test substance is compared tothe same system in which no test substance is added (control), and thesetwo values are compared.

3) The test substance having a higher calcium receptor-stimulatingactivity when the test substance is added is chosen.

4) The kokumi-imparting effect of the chosen test substance is measured,and a test substance having a kokumi-imparting effect is chosen.

The calcium receptor activity is measured by using, for example, ameasurement system using cells expressing the calcium receptor. Thecells may be cells endogenously expressing the calcium receptor, orrecombinant cells which have been transformed with a foreign calciumreceptor gene. As the aforementioned calcium receptor activitymeasurement system, any system may be used without particular limitationso long as the chosen system is able to detect when an extracellularligand (activator) specific to the calcium receptor is added to thecells expressing the calcium receptor, and the activator binds (reactswith) to the calcium receptor, or a detectable signal is transmittedinto the cells in response to binding (reaction) of the activator andthe calcium receptor. When the reaction of the tested compound resultsin calcium receptor activity, this tested compound is determined to havecalcium receptor activation activity and be a kokumi-imparting compound.

The kokumi-imparting effect can be confirmed by a taste test by a human,or the like. Furthermore, the test substance used in the screeningmethod is not particularly limited, and may include low molecular weightcompounds, saccharides, peptides, proteins, and so forth.

The human calcium receptor encoded by the human calcium receptor gene(GenBank Accession No. NM_000388) is preferred. However, the chosencalcium receptor is not limited to the protein encoded by the gene ofthe aforementioned reported GenBank sequence, but it may also be aprotein encoded by a gene having a homology of 60% or more, preferably80% or more, more preferably 90% or more, to the aforementionedsequence, so long as the protein functions as a calcium receptor. TheGPRC6A receptor and the 5.24 receptor are also known to be subtypes ofthe calcium receptor, and they can also be used in the screening methoddescribed herein. The calcium receptor function can be examined byexpressing a gene of interest in a cell and measuring changes in theelectric current, or intracellular calcium ion concentration at the timeof the addition of calcium.

The origin of the calcium receptor is not particularly limited, andexamples include, besides the aforementioned human calcium receptor,calcium receptors derived from animals such as mouse, rat, and dog.

As described above, the calcium receptor activity can be confirmed byusing live cells which express a calcium receptor or a fragment thereof,cell membranes which express a calcium receptor or a fragment thereof,an in vitro system containing a calcium receptor or a fragment thereof,or the like.

An example using live cells is shown below. However, confirmation of thecalcium receptor activity is not limited to this example.

The calcium receptor can be expressed in cultured cells such as Xenopuslaevis oocytes, hamster ovarian cells, and human fetal kidney cells. Thecalcium receptor can be expressed by cloning a calcium receptor gene ina plasmid that contains a foreign gene and introducing the plasmid orcRNA obtained by using the plasmid as a template. To detect thereaction, electrophysiological techniques, fluorescent indicatorreagents that indicate elevation of intracellular calcium level, and soforth, can be used.

Expression of the calcium receptor is first confirmed based on theresponse to calcium or a specific activator. Oocytes can be used thathave an intracellular current with calcium at a concentration of about 5mM, or cultured cells can be used that showed fluorescence of thefluorescent indicator reagent with calcium at a concentration of about 5mM. Calcium concentration dependency is determined by changing thecalcium concentration. Then, a test substance such as a peptide isprepared to a concentration of about 1 μM to 1 mM, and added to theoocytes or cultured cells, and the calcium receptor activity of the testsubstance such as the aforementioned peptide is measured.

Kokumi-Imparting Agent

The compositions described herein include those containing akokumi-imparting substance which is obtained by the screening method.The kokumi-imparting substance is typically the active ingredient in thecomposition. The composition may contain, for example, one or more ofthe following substances: γ-Glu-X-Gly, wherein X represents an aminoacid or amino acid derivative except for Cys, γ-Glu-Val-Y, wherein Yrepresents an amino acid or amino acid derivative, γ-Glu-Ala, γ-Glu-Gly,γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly,Cys-Met, Glu-Cys, Gly-Cys, Leu-Asp, D-Cys, γ-Glu-Met(O),γ-Glu-γ-Glu-Val, γ-Glu-Val-NH₂, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau,γ-Glu-Cys(S-Me)(O), γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, andγ-Glu-Cys(S-Me). These substances may also be referred to as “peptidesand amino acids used for the present invention”. These peptides andamino acids can also be obtained by the screening method describedabove. Here, “amino acid” means, but is not limited to, neutral aminoacids such as Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Asn, Gln,Pro, and Hyp, acidic amino acids such as Asp and Glu, basic amino acidssuch as Lys, Arg, and His, aromatic amino acids such as Phe, Tyr, Trp,and other amino acids such as Homoserine, Citrulline, Ornithine,alpha-Aminobutylic acid, Norvaline, Norleucine and Taurine.

The abbreviations for the amino acid residues are as follows:

(1) Gly: Glycine

(2) Ala: Alanine

(3) Val: Valine

(4) Leu: Leucine

(5) Ile: Isoleucine

(6) Met: Methionine

(7) Phe: Phenylalanine

(8) Tyr: Tyrosine

(9) Trp: Trptophan

(10) His: Histidine

(11) Lys: Lysine

(12) Arg: Arginine

(13) Ser: Serine

(14) Thr: Threonine

(15) Asp: Aspartic acid

(16) Glu: Glutamic acid

(17) Asn: Aspargine

(18) Gln: Glutamine

(19) Cys: Cysteine

(20) Pro: Proline

(21) Orn: Ornithine

(22) Sar: Sarcosine

(23) Cit: Citruline

(24) N-Val: Norvaline

(25) N-Leu: Norleucine

(26) Abu: alpha-Aminobutylic acid

(27) Tau: Taurine

(28) Hyp: Hydroxyproline

(29) t-Leu: tert-leucine

Furthermore, “amino acid derivative” means various types of derivativesof the above-mentioned amino acids, and may include, but are not limitedto, unusual amino acids, non-natural amino acids, amino alcohols,substituted amino acids wherein an amino acid side chain, such ascarbonyl group, amino group, and/or thiol group, is substituted withvarious substituents. Such substituents include an alkyl group, acylgroup, hydroxyl group, amino group, alkylamino group, nitro group,sulfonyl group, and various protection groups. Such substituted aminoacids include, for example, Arg (NO₂): N-γ-nitro arginine, Cys (SNO):S-nitrocysteine, Cys (S-Me): S-methyl cysteine, Cys (S-allyl): S-allylcysteine, Val-NH₂: valinamide, Val-ol: valinol(2-amino-3-methyl-1-butanol).

The “O” in the formulas γ-Glu-Met(O) and γ-Glu-Cys(S-Me)(O) indicates asulfoxide structure. The “γ” in the formula γ-Glu indicates thatglutamic acid bonds to another amino acid via the γ position of thecarboxy group in the glutamic acid. γ-Glu-Cys(SNO)-Gly has the followingstructural formula:

The following peptides have been found to impart kokumi and can be usedin the compositions described herein: γ-Glu-X-Gly, wherein the Xrepresents an amino acid or amino acid derivative except for Cys,γ-Glu-Val-Y, wherein the Y represents an amino acid or amino acidderivative, γ-Glu-Ala, γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr,γ-Glu-Val, γ-Glu-Orn, Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys, Gly-Cys,Leu-Asp, D-Cys, γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH₂,γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu,γ-Glu-Ile, γ-Glu-t-Leu, and γ-Glu-Cys(S-Me). These peptides and aminoacids may be used alone, or in various combinations of two or more.Among these, compounds having a structural formula: γ-Glu-X-Gly, whereinX represents Cys(SNO), Cys(S-allyl), Gly, Cys(S-Me), Abu, or Ser), orγ-Glu-Val-Y, wherein Y represents Gly, Val, Glu, Lys, Phe, Ser, Pro,Arg, Asp, Met, Thr, His, Orn, Asn, Cys, or Gln are preferred.

Among these, the following compounds are novel substances newlysynthesized: γ-Glu-X-Gly, wherein X represents Asn, Gln, His, Lys, Ornor Arg, and γ-Glu-Val-Y, wherein Y represents Leu, Ile, Ser, Thr, Met,Cys, Asp, Asn, Gln, Lys, Orn, Arg, Phe, Tyr, Pro, Hyp, Trp, H is, orAbu. Furthermore, among these novel substances, γ-Glu-X-Gly, wherein Xrepresents Asn, Gln, His, Lys, Orn or Arg and γ-Glu-Val-Y, wherein Yrepresents Ser, Thr, Met, Cys, Asp, Asn, Gln, Lys, Orn, Arg, Pro or Hisare preferred.

Although threshold concentrations, or the minimum concentrations whichallow the sensing of taste, of known taste-imparting peptides are about0.2% ( 1/10 of the threshold concentration of MSG), and thus theirpracticality is poor (J. Agr. Food Chem., vol. 23, No. 1, 49-53 (1975)),the compounds of the present invention show kokumi enhancing activity atan extremely low concentration of about 0.0001 to 0.1%, and thus theyare extremely useful compounds due to their extremely high activity.

Commercially available peptides and amino acids, if available, can beused in the methods and compositions described herein. Furthermore, thepeptides can be obtained by using a known technique such as chemicalsynthesis, or synthesis via an enzymatic reaction. Chemical synthesis isconvenient since the number of amino acid residues of the peptides issmall, for example, 2 or 3 residues. A peptide synthesizer can be usedwhen chemically synthesizing the peptides, either entirely or partially.Examples of such methods include, for example, a peptide solid phasesynthetic method. Peptides synthesized as described above can bepurified by usual means, for example, ion exchange chromatography,reversed phase high performance liquid chromatography, affinitychromatography, and so forth. These peptide solid phase syntheticmethods and the following peptide purification are well known in thistechnical field.

Furthermore, the peptides can also be prepared by an enzymatic reaction.For example, the method described in International Patent PublicationWO2004/011653 can be used. That is, one amino acid or dipeptide with anesterified or amidated carboxyl terminus can be reacted with an aminoacid having a free amino group, for example, an amino acid with aprotected carboxyl group, in the presence of a peptide producing enzyme,and purifying the produced dipeptide or tripeptide. Examples of thepeptide producing enzyme include a culture of microorganisms having anability to produce peptides, microbial cells separated from suchculture, processed products of these cells, peptide producing enzymesderived from such microorganisms, and so forth.

Salt forms of the peptides and amino acids are also included.Pharmacologically acceptable salts may be used for the salt forms of thepeptides and amino acids. Examples of salts containing an acidic groupsuch as a carboxyl group include ammonium salts, salts with alkalimetals such as sodium and potassium, salt with alkaline earth metalssuch as calcium and magnesium, aluminum salts, zinc salts, salts withorganic amines such as triethylamine, ethanolamine, morpholine,pyrrolidine, piperidine, piperazine, and dicyclohexylamine, and saltswith basic amino acids such as arginine and lysine. Examples of saltswith a basic group include salts with inorganic acids such ashydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, andhydrobromic acid, salts with organic carboxylic acids such as aceticacid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaricacid, succinic acid, tannic acid, butyric acid, hibenzoic acid, pamoicacid, enanthoic acid, decanoic acid, teoclic acid, salicylic acid,lactic acid, oxalic acid, mandelic acid, and malic acid, and salts withorganic sulfonic acids such as methanesulfonic acid, benzenesulfonicacid, and p-toluenesulfonic acid.

The method for using the kokumi-imparting substances obtained by thescreening method described herein, and the compositions containing oneor more of these substances is not particularly limited. For example,they can be added to food, seasonings, or drinks, either alone or incombination with other various additives, etc.

Furthermore, the kokumi-imparting agent may be employed alone, or incombination with other known compounds having kokumi-imparting activity,such as glutathione and alliin, or other various additives etc., whichmay be arbitrarily added. Moreover, the kokumi-imparting agent maycontain one or more known compounds having calcium receptor activationactivity.

Examples of these known compounds having calcium receptor activationactivity include cations such as calcium and gadolinium cations, basicpeptides such as polyarginine and polylysine, polyamines such asputrescine, spermine, and spermidine, proteins such as protamine, aminoacids such as phenylalanine and glutathione, cinacalcet, and so forth.Salt forms of these compounds are also included. It has been found thatglutathione has calcium receptor activation activity.

Also, it has been found that the kokumi-imparting activities ofcompounds having known kokumi-imparting activity, such as glutathione,as well as the kokumi-imparting agents described herein are alsoimproved when formulated into a composition with compounds havingcalcium receptor activation activity.

Any known additives typically mixed with food, seasonings, or drink canbe used without particular limitation. Examples of such additivesinclude, for example, perfumes, saccharides, sweetners, dietary fibers,vitamins, amino acids such as sodium glutamate (MSG), nucleic acids suchas inosine monophosphate (IMP), inorganic salts such as sodium chloride,water, and so forth.

The amount of the kokumi-imparting substance, agent, or compositionwhich is obtained by the screening method described herein, may beemployed in an amount effective for imparting kokumi, and can besuitably adjusted depending on the purpose. However, for seasoning,food, or drink, for example, it may be 1 mass ppb to 99.9 mass %,preferably 10 mass ppb to 99.9 mass %, more preferably 10 mass ppm to 10mass % with respect to the seasoning, foodstuff, or drink, in terms ofthe total amount of the kokumi-imparting substance, agent, orcomposition.

Therefore, by adding one or more of the kokumi-imparting substances,agents, or compositions obtained by the screening method describedherein, to food or drink so that the food or drink containsapproximately 1 mass ppb to 99.9 mass %, preferably 10 mass ppb to 99.9mass %, more preferably 10 mass ppm to 10 mass % of the substances oragents, food or drink imparted with kokumi can be produced.

Furthermore, food or drink imparted with kokumi can also be prepared byadding a seasoning containing 1 mass ppb to 99.9 mass % of one or moreof the kokumi-imparting substances obtained by the screening methoddescribed herein. The kokumi-imparting agents, or compositions can beadded to food or drink so that the food or drink contains 0.01 to 10mass %, preferably 0.1 to 10 mass %, of the seasoning.

The kokumi-imparting substance obtained by the screening methoddescribed herein, or the compositions described herein may be in theform of a dry powder, paste, solution, or the like, and the physicalproperties thereof are not particularly limited.

EXAMPLES

Hereinafter, the present invention will be more specifically explainedwith reference to the following non-limiting examples.

Example 1 Preparation of Gene (cRNA)

The gene encoding the calcium receptor was prepared as follows. On thebasis of the DNA sequence registered at NCBI (calcium receptor:NM_000388), synthetic oligo DNAs (forward primer (N) and reverse primer(C)) were prepared based on the DNA sequence registered at NCBI for thecalcium receptor (NM_000388), and used for PCR (Table 1, SEQ ID NOS: 1and 2).

TABLE 1 Synthetic oligo DNAs (forward primer (N)and reverse primer (C), h: human) Code Sequence (5′-3′) hCASR_NACTAATACGACTCACTATAGGGACCATGGCATTTTAT AGCTGCTGCTGG hCASR_CTTATGAATTCACTACGTTTTCTGTAACAG

The primers shown in Table 1 (hCASR_N (SEQ ID NO: 1) and hCASR_C (SEQ IDNO: 2)) were synthesized from human kidney cDNA (Clontech), and PCR wasperformed with Pfu ultra DNA Polymerase (Stratagene) under the followingconditions: after a reaction at 94° C. for 3 minutes, a cycle ofreactions at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 2 minutes was repeated 35 times, and then a reaction was performedat 72° C. for 7 minutes. Whether amplification was attained by PCR wasdetected by agarose electrophoresis, staining with a DNA stainingreagent, and ultraviolet irradiation. The length of the PCR productswere confirmed by comparison with DNA markers of known sizessimultaneously subjected to electrophoresis. The plasmid vector pBR322(Takara) was digested with the restriction enzyme EcoRV. The genefragment amplified by PCR was ligated to the cleavage site of theplasmid by using Ligation Kit (Promega). The Escherichia coli DH5αstrain was transformed with each ligation reaction solution, and thetransformants containing the plasmid with the PCR amplification productwere cloned was selected. The PCR amplification product was confirmed byDNA sequence analysis. By using this recombinant plasmid as a templatetogether with a cRNA preparation kit (Ambion), the cRNA of the calciumreceptor gene was prepared.

Example 2 Preparation of Various Samples

23 special grade amino acids were employed, including alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, ornithine,taurine (all of these from Ajinomoto), and hydroxyproline (NakaraiTesque). Special grade amino acids were also used as D-Cys and D-Trp(Nakarai Tesque) and calcium chloride. Furthermore, the followingpeptide samples were used: γ-Glu-Cys-Gly (Sigma Aldrich Japan),γ-Glu-Cys(SNO)-Gly (Dojin Chemical Laboratory), γ-Glu-Ala (BachemFeinchemikalien AG), γ-Glu-Gly (Bachem Feinchemikalien AG), γ-Glu-Cys(Sigma Aldrich Japan), γ-Glu-Met (Bachem Feinchemikalien AG),γ-Glu-Abu-Gly (Abu: α-aminobutyric acid, Bachem Feinchemikalien AG),γ-Glu-Thr (Kokusan Chemical), γ-Glu-Val (Kokusan Chemical), γ-Glu-Leu(contract manufactured product), γ-Glu-Ile (contract manufacturedproduct), γ-Glu-Orn (Kokusan Chemical), Asp-Gly (contract manufacturedproduct), Cys-Gly (contract manufactured product), Cys-Met (contractmanufactured product), Glu-Cys (contract manufactured product), Gly-Cys(contract manufactured product), Leu-Asp (contract manufacturedproduct), γ-Glu-Val-Val (contract manufactured product), γ-Glu-Val-Glu(contract manufactured product), γ-Glu-Val-Lys (contract manufacturedproduct), γ-Glu-γ-Glu-Val (contract manufactured product), γ-Glu-Gly-Gly(contract manufactured product), γ-Glu-Val-Phe (contract manufacturedproduct), γ-Glu-Val-Ser (contract manufactured product), γ-Glu-Val-Pro(contract manufactured product) γ-Glu-Val-Arg (contract manufacturedproduct), γ-Glu-Val-Asp (contract manufactured product),γ-Glu-Val-Met(contract manufactured product), γ-Glu-Val-Thr (contractmanufactured product), γ-Glu-Val-His(contract manufactured product),γ-Glu-Val-Asn (contract manufactured product), γ-Glu-Val-Gln (contractmanufactured product), γ-Glu-Val-Cys(contract manufactured product),γ-Glu-Val-Orn (contract manufactured product) and γ-Glu-Ser-Gly(contract manufactured product). Glutamine and cysteine were preparedupon use, and the other samples were stored at 20° C. after preparation.Peptides having a purity of 90% or higher were used, except forγ-Glu-Cys, which was at a purity of 80% or higher. The pH was adjusted,as needed, to an approximately neutral pH with NaOH or HCl. The solutionused for dissolution of amino acids and peptides, preparation of Xenopuslaevis oocytes, and culture of the oocytes had the followingcomposition: 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM Hepes,pH 7.2.

Example 3 Synthesis of γ-Glu-Val-Gly

Boc-Val-OH (8.69 g, 40.0 mmol) and Gly-OBzl.HCl (8.07 g, 40.0 mmol) weredissolved in methylene chloride (100 ml), and the solution wasmaintained at 0° C. Triethylamine (6.13 ml, 44.0 mmol), HOBt(1-hydroxybenzotriazole, 6.74 g, 44.0 mmol), and WSC.HCl(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 8.44 g,44.0 mmol) were added to the solution, and the mixture was stirredovernight at room temperature. The reaction mixture was concentratedunder reduced pressure, and the residue was dissolved in ethyl acetate(200 ml). The solution was washed with water (50 ml), 5% citric acidaqueous solution (50 ml× twice), saturated brine (50 ml), 5% sodiumhydrogencarbonate aqueous solution (50 ml× twice), and saturated brine(50 ml). The organic layer was dried over anhydrous magnesium sulfate,then the magnesium sulfate was removed by filtration, and the filtratewas concentrated under reduced pressure. The residue was recrystallizedfrom ethyl acetate/n-hexane to obtain white crystals of Boc-Val-Gly-OBzl(13.2 g, 36.2 mmol).

Boc-Val-Gly-OBzl (5.47 g, 15.0 mmol) was added to 4 N HCl/dioxanesolution (40 ml), and the mixture was stirred at room temperature for 50minutes. Dioxane was removed by concentration under reduced pressure,n-hexane (30 ml) was added to the residue, and the mixture wasconcentrated under reduced pressure. This procedure was repeated 3 timesto quantitatively obtain H-Val-Gly-OBzl.HCl.

Then, H-Val-Gly-OBzl.HCl and Z-Glu-OBzl (5.57 g, 15.0 mmol) weredissolved in methylene chloride (50 ml), and the solution was kept at 0°C. Triethylamine (2.30 ml, 16.5 mmol), HOBt (1-hydroxybenzotriazole,2.53 g, 16.5 mmol), and WSC.HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 3.16 g, 16.5 mmol) were added to thesolution, and the mixture was stirred at room temperature for 2 days.The reaction mixture was concentrated under reduced pressure, and theresidue was dissolved in heated ethyl acetate (1500 ml). The solutionwas washed with water (200 ml), 5% citric acid aqueous solution (200 ml×twice), saturated brine (150 ml), 5% sodium hydrogencarbonate aqueoussolution (200 ml× twice), and saturated brine (150 ml). The organiclayer was dried over anhydrous magnesium sulfate, then the magnesiumsulfate was removed by filtration, and the filtrate was concentratedunder reduced pressure. The deposited crystals were collected byfiltration, and dried under reduced pressure to obtain white crystals ofZ-Glu(Val-Gly-OBzl)-OBzl (6.51 g, 10.5 mmol).

Then, Z-Glu(Val-Gly-OBzl)-OBzl (6.20 g, 10.03 mmol) was suspended inethanol (200 ml), and 10% palladium/carbon (1.50 g) was added. Areduction reaction was performed at 55° C. for 5 hours under a hydrogenatmosphere. During the reaction, a total amount of 100 ml of water wasgradually added. The catalyst was removed by filtration using a Kiriyamafunnel, and the filtrate was concentrated under reduced pressure to ahalf volume. The reaction mixture was further filtered through amembrane filter, and the filtrate was concentrated under reducedpressure. The residue was dissolved in a small volume of water, ethanolwas added to deposit crystals, and the crystals were collected byfiltration and dried under reduced pressure to obtain a white powder ofγ-Glu-Val-Gly (2.85 g, 9.40 mmol).

ESI-MS: (M+H)⁺=304.1

¹H-NMR (400 MHz, D₂O) δ (ppm): 0.87 (3H, d, J=6.8 Hz), 0.88 (3H, d,J=6.8 Hz), 1.99-2.09 (3H, m), 2.38-2.51 (2H, m) 3.72 (1H, t, J=6.35 Hz),3.86 (1H, d, J=17.8 Hz), 3.80 (1H, d, J=17.8 Hz), 4.07 (1H, d, J=6.8 Hz)

Example 4 Synthesis of γ-Glu-Cys(S-Me)-Gly [Cys(S-Me): S-methylcysteine]

Reduced glutathione (15.0 g, 48.8 mmol) was added to water (45 ml), andthen sodium hydroxide (4.52 g, 2.2 equivalents, 107 mmol) was addedlittle by little to the mixture while bubbled with nitrogen. Then,methyl iodide (4.56 ml, 1.5 equivalents, 73 mmol) was added to themixture, and the mixture was sealed and stirred at room temperature for2 hours. The reaction mixture was adjusted to pH 2 to 3 withconcentrated hydrochloric acid, added with ethanol (150 ml), and storedovernight in a refrigerator. Since oily matter separated, thesupernatant was removed. When the remaining oily matter was dissolved inwater and ethanol was gradually added, partially crystallized oilymatter was deposited. Therefore, the supernatant liquid was removedagain. The residue was dissolved in water (300 ml), adsorbed onto an ionexchange resin (Dowex 1-acetate, 400 ml) applied to a column, and afterwashing with water, eluted with 1 N acetic acid aqueous solution. Theeluate was concentrated under reduced pressure, and precipitated withwater/ethanol to obtain a white powder of γ-Glu-Cys(S-Me)-Gly (5.08 g,15.8 mmol).

FAB-MS: (M+H)⁺=322

¹H-NMR (400 MHz, D₂O) δ (ppm): 2.14 (3H, s), 2.15-2.22 (2H, m),2.50-2.58 (2H, m), 2.86 (1H, dd, J=9.0 Hz, J=14.0 Hz), 3.03 (1H, dd,J=5.0 Hz, J=14.0 Hz), 3.84 (1H, t, J=6.5 Hz), 3.99 (2H, S), 4.59 (1H,dd, J=5.0 Hz, J=9.0 Hz)

Example 5 Synthesis of Other Peptides

γ-Glu-Met(O), γ-Glu-Val-NH₂, γ-Glu-Val-ol, γ-Glu-Ser, γ-Glu-Tau,γ-Glu-Cys(S-Me)(O), γ-Glu-t-Leu, γ-Glu-Cys(S-allyl)-Gly, andγ-Glu-Cys(S-Me) were synthesized in a manner similar to that describedin Examples 3 and 4.

Example 6 Evaluation of Calcium Receptor Activation Activity

To evaluate calcium receptor activation activity, a Ca ionconcentration-dependent Cl ionic current measuring method using aXenopus laevis oocyte expression system was used. If each activator isadded to Xenopus laevis oocytes expressing the calcium receptor,intracellular Ca ions increase. Then, the Ca ion concentration-dependentCl channel opens, and the intracellular current value changes as anionic current. By measuring the change in this intracellular currentvalue, the presence or absence of calcium receptor activation activitycan be determined.

Specifically, the abdomen of Xenopus laevis was opened, and an egg batchwas taken out and treated with a 1% collagenase solution at 20° C. for 2hours to obtain individual oocytes. Into the oocytes, 50 nl of 1 μg/μlreceptor cRNA or 50 nl of sterilized water per oocyte was introduced byusing a micro glass capillary, and the oocytes were cultured at 18° C.for 2 or 3 days. For the culture, a solution obtained by adding 2 mMpyruvic acid, 10 U/ml of penicillin, and 10 μg/ml of streptomycin to thesolution in Example 2 was used. After the culture, a test solution wasadded to the oocytes containing either the cRNA or sterilized water.Electrophysiological measurement was performed by using an amplifier,Geneclamp500 (Axon), and recording software, AxoScope 9.0 (Axon). Theoocytes were voltage-clamped at −70 mV by the double electrode voltageclamp method, and the intracellular current was measured via the Ca ionconcentration-dependent Cl ion channel. The maximum value of theintracellular current was considered as the response current value.

Example 7 Evaluation of Calcium Receptor Activation Activity of Calcium

The calcium receptor activation activity of calcium was evaluated byusing the method described in Example 6. That is, oocytes containingeither cRNA of the calcium receptor or sterilized water were prepared,and voltage-clamped at −70 mV by the double electrode voltage clampmethod. To the voltage-clamped oocytes, calcium was added (2 mM, 5 mM,10 mM, 20 mM), and Ca ion concentration-dependent Cl response currentwas measured. The results are shown in FIG. 1. From these results, itwas confirmed that the cRNA of the calcium receptor was functionallyexpressed in the oocytes. Furthermore, since the oocytes containingwater did not respond to even high concentration calcium, it wasconfirmed that the calcium receptor is not expressed in the oocytes.

Example 8 Evaluation of Calcium Receptor Activation Activity of L-AminoAcids

Calcium receptor activation activity of L-amino acids was evaluated byusing the method described in Example 6. That is, oocytes containingeither cRNA of the calcium receptor or sterilized water were prepared,and voltage-clamped at −70 mV by the double electrode voltage clampmethod. To the voltage-clamped oocytes, alanine (10 mM), arginine (10mM), asparagine (10 mM), aspartic acid (10 mM), cysteine (10 mM),glutamine (10 mM), glutamic acid (10 mM), glycine (10 mM), histidine (10mM), isoleucine (10 mM), leucine (10 mM), lysine (10 mM), methionine (10mM), phenylalanine (10 mM), proline (10 mM), serine (10 mM), threonine(10 mM), tryptophan (10 mM), tyrosine (10 mM), valine (10 mM), ornithine(10 mM), taurine (10 mM), or hydroxyproline (10 mM) was added, and Caion concentration-dependent Cl response current was measured. Theresults are shown in FIG. 2. By these results, it was demonstrated thatcysteine, histidine, phenylalanine, tryptophan, and tyrosine haddefinite calcium receptor activation activity. As for the aforementionedamino acids, the activation activity was reported in Proc. Natl. Acad.Sci. USA, 2000 Apr. 25, 97(9):4814-9.

Example 9 Evaluation of Calcium Receptor Activation Activity ofD-Cysteine

Calcium receptor activation activity of D-cysteine was evaluated byusing the method described in Example 6. That is, oocytes containingeither cRNA of the calcium receptor or sterilized water were prepared,and voltage-clamped at −70 mV by the double electrode voltage clampmethod. To the voltage-clamped oocytes, D-cysteine (10 mM), L-cysteine(10 mM), D-tryptophan (10 mM), or L-tryptophan (10 mM) was added, and Caion concentration-dependent Cl response current was measured. Theresults are shown in FIG. 3. By these results, it was demonstrated thatD-cysteine had definitive calcium receptor activation activity.

Example 10 Evaluation of Calcium Receptor Activation Activity ofPeptides

Calcium receptor activation activity of peptides was evaluated by usingthe method described in Example 6. That is, oocytes containing eithercRNA of the calcium receptor or sterilized water were prepared, andvoltage-clamped at −70 mV by the double electrode voltage clamp method.To the voltage-clamped oocytes, γ-Glu-Cys-Gly (50 μM),γ-Glu-Cys(SNO)-Gly (50 μM), γ-Glu-Ala (50 μM), γ-Glu-Gly (500 μM),γ-Glu-Cys (50 μM), γ-Glu-Met (500 μM), γ-Glu-Thr (50 μM), γ-Glu-Val (50μM), γ-Glu-Orn (500 μM), Asp-Gly (1 mM), Cys-Gly (1 mM), Cys-Met (1 mM),Glu-Cys (50 μM), Gly-Cys (500 μM), or Leu-Asp (1 mM) was added, and Caion concentration-dependent Cl response current was measured. Theresults are shown in FIG. 4. By these results, it was demonstrated thatthe aforementioned peptides had definitive calcium receptor activationactivity.

Example 11 Evaluation of Calcium Receptor Activation Activity ofPeptides

Calcium receptor activation activity of peptides was evaluated in thesame manner as that of Example 10. Each of the peptides shown in Table 2was added to voltage-clamped oocytes at 1000 μM, 300 μM, 100 μM, 30 μM,10 μM, 3 μM, 1 μM, 0.3 μM, and 0.1 μM, and Ca ionconcentration-dependent Cl response current was measured. The lowestconcentration for which current was detected was shown in Table 2 as theactivity. From these results, it became clear that these 32 peptides hadcalcium receptor activation activity.

TABLE 2 No. Peptide Activity 1 γ-Glu-Met(O) 1000 μM 2 γ-Glu-Val-Val 1000μM 3 γ-Glu-Val-Glu 1000 μM 4 γ-Glu-Val-Lys 1000 μM 5 γ-Glu-Val-Arg 1000μM 6 γ-Glu-Val-Asp 1000 μM 7 γ-Glu-Val-Met 1000 μM 8 γ-Glu-Val-Thr 1000μM 9 γ-Glu-γ-Glu-Val 1000 μM 10 γ-Glu-Val-NH2 1000 μM 11 γ-Glu-Val-ol1000 μM 12 γ-Glu-Ser 300 μM 13 γ-Glu-Tau 300 μM 14 γ-Glu-Cys(S—Me)(O)300 μM 15 γ-Glu-Val-His 100 μM 16 γ-Glu-Val-Orn 100 μM 17 γ-Glu-Leu 100μM 18 γ-Glu-Ile 100 μM 19 γ-Glu-t-Leu 100 μM 20 γ-Glu-Cys(S-allyl)-Gly100 μM 21 γ-Glu-Val-Asn 30 μM 22 γ-Glu-Gly-Gly 30 μM 23 γ-Glu-Val-Phe 30μM 24 γ-Glu-Val-Ser 30 μM 25 γ-Glu-Val-Pro 30 μM 26 γ-Glu-Ser-Gly 30 μM27 γ-Glu-Cys(S—Me) 30 μM 28 γ-Glu-Val-Cys 10 μM 29 γ-Glu-Val-Gln 10 μM30 γ-Glu-Abu-Gly 3 μM 31 γ-Glu-Cys(S—Me)-Gly 3 μM 32 γ-Glu-Val-Gly 0.1μM

Example 12 Kokumi-Imparting Activity of Peptides and Amino Acids

Calcium receptor activation activity was confirmed for the followingpeptides and amino acids: γ-Glu-X-Gly (X is Cys(SNO), Cys(S-allyl), Gly,Cys(S-Me), Abu, or Ser), γ-Glu-Val-Y (Y is Gly, Val, Glu, Lys, Phe, Ser,Pro, Arg, Asp, Met, Thr, His, Orn, Asn, Cys, or Gln), γ-Glu-Ala,γ-Glu-Gly, γ-Glu-Cys, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val, γ-Glu-Orn,Asp-Gly, Cys-Gly, Cys-Met, Glu-Cys, Gly-Cys, Leu-Asp, D-Cys,γ-Glu-Met(O), γ-Glu-γ-Glu-Val, γ-Glu-Val-NH₂, γ-Glu-Val-ol, γ-Glu-Ser,γ-Glu-Tau, γ-Glu-Cys(S-Me)(O), γ-Glu-Leu, γ-Glu-Ile, γ-Glu-t-Leu, andγ-Glu-Cys(S-Me). Whether or not these peptides and amino acids havekokumi-imparting activity or not was determined by a sensory evaluationtest, performed as follows. Samples of either alliin (S-allyl-cysteinesulfoxide: control for Kokumi-imparting activity), γ-Glu-Cys-Gly,γ-Glu-Cys, γ-Glu-Ala, or γ-Glu-Val were added to a final concentrationof 0.2 g/dl to distilled water containing sodium glutamate (0.05 g/dl),inosine monophosphate (0.05 g/dl), and calcium chloride (1 mM), andkokumi-imparting activity was determined for each mixture. Samplesolutions that became acidic after dissolution of the samples wereadjusted to pH 6.8 to 7.2 with NaOH before use. The results are shown inTable 3.

TABLE 3 Kokumi-imparting activity of calcium receptor activators Calciumreceptor activator Kokumi-imparting activity γ Glu-Cys-Gly + γ Glu-Cys +γ Glu-Ala + γ Glu-Val +

Example 13 Kokumi-Imparting Activity of Peptides

The intensity of the kokumi-imparting activity of each peptide having aconfirmed calcium receptor activation activity was measured by aquantitative sensory evaluation test, performed as follows: Samples ofeither γ-Glu-Cys-Gly (glutathione), γ-Glu-Ala, γ-Glu-Met, or γ-Glu-Valwere added to distilled water (0.1 g/dl) containing sodium glutamate(0.05 g/dl), inosine monophosphate (0.05 g/dl), and sodium chloride (0.5g/dl), and the intensity of the kokumi-imparting activity was measured.Sample solutions that became acidic after dissolution of the sampleswere adjusted to pH 6.8 to 7.2 with NaOH before use. Sensory evaluationscores were used for evaluation based on the control sample (0 points)and the glutathione sample (3 points), and the test was performed withn=3. The results are shown in Table 4.

TABLE 4 Intensity of kokumi First and Concentration middle Sample (g/dl)taste Aftertaste Evaluation remarks Control — 0 0 — γ-Glu-Cys- 0.1 3.03.0 Thickness, growth Gly (mouthfulness), and continuity were enhanced.γ-Glu-Ala 0.1 0.5 0.2 Although the effect was weak, thickness wasslightly enhanced. γ-Glu-Met 0.1 1.5 0.4 Thickness, and growth(mouthfulness) were slightly enhanced. γ-Glu-Val 0.1 3.0 1.0 Thickness,and growth (mouthfulness) were enhanced mainly for first and middletastes

Example 14 Kokumi-Imparting Activity of Peptides

The intensity of the kokumi-imparting activity of each peptide havingconfirmed calcium receptor activation activity was measured by aquantitative sensory evaluation test, performed as follows: Samples ofeither γ-Glu-Cys-Gly (glutathione), γ-Glu-Cys, γ-Glu-Val, orγ-Glu-Val-Gly were added to distilled water (0.1 g/dl, or 0.01 g/dl asrequired) containing sodium glutamate (0.05 g/dl), inosine monophosphate(0.05 g/dl), and sodium chloride (0.5 g/dl), was, and the intensity ofthe kokumi-imparting activity was measured. The sample solutions thatbecame acidic after dissolution of the samples were adjusted to pH 6.8to 7.2 with NaOH before use. Sensory evaluation scores were used forevaluation based on the control sample (0 points) and the glutathionesample (3 points), and the test was performed with n=5. The results areshown in Table 5.

TABLE 5 Intensity of kokumi First and Concentration middle Sample (g/dl)taste Aftertaste Evaluation remarks Control — 0 0 — γ-Glu-Cys- 0.1 3.03.0 Thickness, growth Gly (mouthfulness), and continuity were enhanced.γ-Glu-Cys 0.1 2.0 2.0 The effect was slightly weaker, but substantiallyequivalent compared with γGlu-Cys-Gly γ-Glu-Val 0.1 3.0 1.0 Thickness,and growth (mouthfulness) were enhanced mainly for first and middletastes γ-Glu-Val- 0.1 * * * Gly γ-Glu-Val- 0.01 3.0 3.0 Thickness, andcontinuity were Gly mainly enhanced. Total taste was enhanced *Unmeasurable: Kokumi-imparting activity was too strong and could not bemeasured by the sensory evaluation.

Example 15 Kokumi-Imparting Activity of Peptides

The intensity of the kokumi-imparting activity of each peptide havingconfirmed calcium receptor activation activity was measured by aquantitative sensory evaluation test, performed as follows: Samples ofeither γ-Glu-Cys-Gly (glutathione), γ-Glu-Abu-Gly, or γ-Glu-Val-Gly wereadded to distilled water (0.1 g/dl or 0.01 g/dl) containing sodiumglutamate (0.05 g/dl), inosine monophosphate (0.05 g/dl), and sodiumchloride (0.5 g/dl), and the intensity of the kokumi-imparting activitywas measured. Sample solutions that became acidic after dissolution ofthe samples were adjusted to pH 6.8 to 7.2 with NaOH before use. Sensoryevaluation scores were used for evaluation based on the control sample(0 points) and the glutathione sample (3 points), and the test wasperformed with n=12. The results are shown in Table 6.

TABLE 6 Intensity of kokumi Concentration First and Sample (g/dl) middletaste Aftertaste Evaluation remarks Control — 0 0 — γGlu-Cys-Gly 0.1 3.03.0 Thickness, growth (mouthfulness), and continuity were enhanced.γGlu-Abu-Gly 0.01 3.0 2.0 Thickness, and growth (mouthfulness) wereenhanced mainly for first and middle tastes. γGlu-Val-Gly 0.01 3.0 3.0Thickness, and continuity were mainly enhanced. Total taste wasenhanced.

Example 16 Activity of Peptides on Basic Tastes

The intensity of the activity on basic tastes of each peptide havingconfirmed calcium receptor activation activity was measured by aquantitative sensory evaluation test, performed as follows: Samples ofeither γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly were added todistilled water (0.0001 to 1 g/dl) containing sodium glutamate (0.2g/dl) for the umami standard, sucrose (5 g/dl) for the sweet tastestandard, sodium chloride (0.7 g/dl) or the salty taste standard, orcitric acid (0.05 g/dl) for the sour taste standard, and the intensityof the activity on basic tastes was measured for each sample.

Sample solutions that became acidic after dissolution of the sampleswith respect to the standard solutions without the samples were adjustedwith NaOH to pH not lower or higher by 0.2 than pH of the standardsolutions before use. Sensory evaluation scores were used to evaluatethe intensity as follows: 0 points for the control sample, 1 point forfairly intense activity as compared to the control, and 2 points forintense activity as compared to the control, and the test was performedwith n=12. The samples showed that the basic tastes were enhanced atconcentrations within the aforementioned broad concentration range. Theresults for typical concentrations are shown in Table 7.

TABLE 7 γGlu- Evaluation Distilled Cys-Gly γGlu-Val-Gly γGlu-Val-Glysystem water 0.10 g/dl 0.005 g/dl 0.01 g/dl Umami 0 0.7 0.7 1.5 Sweettaste 0 1.5 0.5 1.0 Salty taste 0 0.2 0.5 1.0 Sour taste 0 1.5 0.5 1.0

Example 17 Activity of Peptides for Imparting Kokumi to Consomme Soup

The intensity of activity for imparting kokumi to consomme soup of eachpeptide having confirmed calcium receptor activation activity wasmeasured by a quantitative sensory evaluation test, performed asfollows: Consomme soup was prepared by dissolving consomme soup powder(35% of sodium chloride, 18% of sodium glutamate, 0.2% of inosinemonophosphate, 0.3% of white pepper powder, 0.5% of black pepper powder,8.0% of beef extract powder, 3.0% of white wine powder, 2.0% of celerypowder, 8.0% of Chinese cabbage extract powder, 2.5% of onion extractpowder, 25.5% of lactose) at a concentration of 5 g/dl. To this consommesoup, samples of either γ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly wasadded to a concentration of 0.0001 to 1 g/dl, and the intensity of thekokumi-imparting activity was measured for each sample. The consommesoup with the samples that became acidic after dissolution of thesamples with respect to the consomme soup without the samples wasadjusted with NaOH to pH not lower or higher by 0.2 than pH of theconsomme soup without the samples before use. Sensory evaluation scoreswere used for evaluation as follows: 0 points for the control sample, 3points for intense activity as compared to the control, and 5 points forextremely intense activity as compared to the control, and the test wasperformed with n=12. The samples showed kokumi-imparting activity atconcentrations within the aforementioned broad concentration range. Theresults for typical concentrations are shown in Table 8.

TABLE 8 Intensity of kokumi Concentration First and Sample (g/dl) middletaste Aftertaste Evaluation remarks Control — 0 0 — γGlu-Cys-Gly 0.013.0 3.0 Thickness, growth (mouthfulness), and continuity were enhanced.γGlu-Val-Gly 0.0005 2.5 3.0 Thickness and growth (mouthfulness) weremainly enhanced from first and middle taste. γGlu-Val-Gly 0.001 3.0 3.5Thickness and growth (mouthfulness) were mainly enhanced from first andmiddle taste. γGlu-Val-Gly 0.01 5.0 5.0 Thickness and continuity weremainly enhanced. Total taste was enhanced. γGlu-Val-Gly 0.1 5.0 5.0Thickness and continuity were mainly enhanced. Total taste was enhanced.

Example 18 Activity of Peptides for Imparting Kokumi to Japanese ClearSoup

The intensity of activity for imparting kokumi to clear soup of eachpeptide having confirmed calcium receptor activation activity wasmeasured by a quantitative sensory evaluation test, performed asfollows: Japanese clear soup was prepared by adding 0.5 g/dl of soysauce and 0.6 g/dl of sodium chloride to bonito kelp stock, obtained byadding 5 g of dried kelp to 3 L of water, heating the water, adding 25 gof dried bonito flakes just before boiling, and then filtering the watercontaining kelp and bonito flakes. To this clear soup, samples of eitherγ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly was added to aconcentration of 0.0001 to 1 g/dl, and the intensity of thekokumi-imparting activity was measured for each sample. The clear soupwith the samples that became acidic after dissolution of the sampleswith respect to the clear soup without the samples were adjusted withNaOH to pH not lower or higher by 0.2 than pH of the clear soup withoutthe samples before use. Sensory evaluation scores were used to evaluateas follows: 0 points for the control sample, 3 points for intenseactivity as compared to the control, and 5 points for extremely intenseactivity as compared to the control, and the test was performed withn=12. The samples showed kokumi-imparting activity at concentrationswithin the aforementioned broad concentration range. The results fortypical concentrations are shown in Table 9.

TABLE 9 Intensity of kokumi Concentration First and Sample (g/dl) middletaste Aftertaste Evaluation remarks Control — 0 0 — γGlu-Cys-Gly 0.012.0 2.0 Thickness and growth (mouthfulness) were enhanced. γGlu-Val-Gly0.0005 2.5 3.0 Thickness, growth (mouthfulness) and continuity weremainly enhanced from first and middle taste. γGlu-Val-Gly 0.001 3.5 4.0Thickness, growth (mouthfulness) and continuity were mainly enhancedfrom first and middle taste. γGlu-Val-Gly 0.01 5.0 5.0 Thickness, andcontinuity were mainly enhanced. Total taste was enhanced. γGlu-Val-Gly0.1 5.0 5.0 Thickness, and continuity were mainly enhanced. Total tastewas enhanced.

Example 19 Activity of Peptides for Imparting Kokumi to Corn Soup

The intensity of the activity for imparting kokumi to corn soup of eachpeptide having confirmed calcium receptor activation activity wasmeasured by a quantitative sensory evaluation test, performed asfollows: to commercially available corn soup, samples of eitherγ-Glu-Cys-Gly (glutathione) or γ-Glu-Val-Gly were added to aconcentration of 0.0001 to 1 g/dl, and the intensity of thekokumi-imparting activity was measured for each sample. The corn soupwith the samples that became acidic after dissolution of the sampleswith respect to the corn soup without the samples was adjusted with NaOHto pH not lower or higher by 0.2 than pH of the corn soup without thesamples before use. Sensory evaluation scores were used to evaluate asfollows: 0 points for the control sample, 3 points for intense activityas compared to the control, and 5 points for extremely intense activityas compared to the control, and the test was performed with n=12. Thesamples showed kokumi-imparting activity at concentrations within theaforementioned broad concentration range. The results for typicalconcentrations are shown in Table 10.

TABLE 10 Intensity of kokumi First Concentration and middle Sample(g/dl) taste Aftertaste Evaluation remarks Control — 0 0 — γGlu-Cys-Gly0.01 3.0 3.0 Richness, thickness and growth (mouthfulness) wereenhanced. γGlu-Val-Gly 0.0005 2.5 3.0 Sweet taste, growth (mouthfulness)and continuity were mainly enhanced from first and middle taste.γGlu-Val-Gly 0.001 3.5 4.0 Sweet taste, growth (mouthfulness) andcontinuity were mainly enhanced from first and middle taste.γGlu-Val-Gly 0.01 4.5 5.0 Growth (mouthfulness) and continuity weremainly enhanced. Total taste was enhanced. γGlu-Val-Gly 0.1 5.0 5.0Growth (mouthfulness) and continuity were mainly enhanced. Total tastewas enhanced.

Example 20 Activity of Peptides for Imparting Kokumi to Curry Sauce

The intensity of the activity for imparting kokumi to curry sauce ofeach peptide having confirmed calcium receptor activation activity wasmeasured by a quantitative sensory evaluation test, performed asfollows: to curry sauce prepared in a conventional manner by usingcommercially available curry roux, samples of either γ-Glu-Cys-Gly(glutathione) or γ-Glu-Val-Gly were added to a concentration of 0.0001to 1 g/dl, and the intensity of kokumi-imparting activity was measuredfor each sample. The curry sauces with the samples that became acidicafter dissolution of the samples with respect to the curry soup withoutthe samples were adjusted with NaOH to pH not lower or higher by 0.2than pH of the curry soup without the samples before use. Sensoryevaluation scores were used to evaluate as follows: 0 points for thecontrol sample, 3 points for intense activity as compared to thecontrol, and 5 points for extremely intense activity as compared to thecontrol, and the test was performed with n=12. The samples showedkokumi-imparting activity at concentrations within the aforementionedbroad concentration range. The results for typical concentrations areshown in Table 11.

TABLE 11 Intensity of kokumi First Concentration and middle Sample(g/dl) taste Aftertaste Evaluation remarks Control — 0 0 — γGlu-Cys-Gly0.01 3.0 3.0 Richness, thickness and continuity were enhanced.γGlu-Val-Gly 0.001 2.5 3.0 Mildness, richness and growth (mouthfulness)were mainly enhanced. γGlu-Val-Gly 0.005 3.5 4.0 Mildness, richness andgrowth (mouthfulness) were mainly enhanced. γGlu-Val-Gly 0.01 5.0 5.0Richness and continuity were mainly enhanced. Total taste was enhanced.γGlu-Val-Gly 0.1 5.0 5.0 Richness and continuity were mainly enhanced.Total taste was enhanced.

Example 21 Kokumi-Imparting Activity Observed when Peptides andAdditives Such as Known Calcium Receptor Activators were Used inCombination

The intensity of the kokumi-imparting activity of each peptide havingconfirmed calcium receptor activation activity and a known calciumreceptor activator used in combination was measured by a quantitativesensory evaluation test, performed as follows: Samples of γ-Glu-Cys-Gly(glutathione) or γ-Glu-Val-Gly (0.0001 to 1 g/dl), or these samplescombined with a calcium receptor activator such as calcium lactate,protamine or polylysine, or GABA (addition concentration: 0.0001 to 1g/dl) were added to distilled water containing sodium glutamate (0.05g/dl), inosine monophosphate (0.05 g/dl), and sodium chloride (0.5g/dl), and the intensity of the kokumi-imparting activity was measured.Sample solutions that became acidic after dissolution of the sampleswere adjusted to pH 6.8 to 7.2 with NaOH before use. Sensory evaluationscores were used to evaluate as follows: 0 points for the controlsample, 3 points for intense activity (as intensity of 0.05 g/dlγ-Glu-Cys-Gly and 0.005 g/dl γ-Glu-Val-Gly), and 6 points for extremelyintense activity (as twice intensity of 0.05 g/dl γ-Glu-Cys-Gly and0.005 g/dl γ-Glu-Val-Gly), and the test was performed with n=12. Thesamples showed kokumi-imparting activity at concentrations within theaforementioned broad concentration range. The results for typicalconcentrations are shown in Table 12. As a result, a compound known tohave kokumi-imparting activity, glutathione, can have improvedkokumi-imparting activity when used with a known calcium receptoractivator or the like such as calcium.

TABLE 12 Intensity of kokumi First and Peptide ConcentrationConcentration middle sample (g/dl) Additive (g/dl) taste AftertasteEvaluation remarks — — — — 0 0 γGlu-Cys- 0.05 — — 3.0 3.0 Thickness,growth Gly (mouthfulness) and continuity γGlu-Val- 0.005 — — 3.0 3.0Thickness and Gly continuity — — Calcium 0.25 0.5 0.5 Thickness lactate— — Protamine 0.005 1.5 1.0 Growth (mouthfulness) — — Polylysine 0.0010.5 0.5 Thickness — — GABA 0.025 0.5 0.5 Thickness γGlu-Cys- 0.05Calcium 0.25 3.5 4.0 Thickness, growth Gly lactate (mouthfulness) andcontinuity γGlu-Cys- 0.05 Protamine 0.005 4.5 4.5 Thickness, growth Gly(mouthfulness) and continuity γGlu-Cys- 0.05 Polylysine 0.001 3.5 4.0Thickness, growth Gly (mouthfulness) and continuity γGlu-Cys- 0.05 GABA0.025 4.5 4.5 Thickness, growth Gly (mouthfulness) and continuityγGlu-Val- 0.005 Calcium 0.25 5.0 5.0 Thickness and Gly lactatecontinuity γGlu-Val- 0.005 Protamine 0.005 4.5 4.5 Thickness, growth Gly(mouthfulness) and continuity γGlu-Val- 0.005 Polylysine 0.001 4.5 4.5Thickness and Gly continuity γGlu-Val- 0.005 GABA 0.025 4.5 4.5 Richnessand Gly thickness

INDUSTRIAL APPLICABILITY

The specific amino acids and peptides having calcium receptor activationactivity are also useful as kokumi-imparting substances. In particular,as shown in Examples 12 to 21, several kinds of dipeptides andtripeptides were newly discovered to be kokumi-imparting substances, andsince they are peptides, they can be used in the field of foodstuffs inwhich high safety is demanded. In addition, since a method for screeningfor a kokumi-imparting substance utilizing calcium receptor activationas an index has been developed, so-called high throughput screening canbe used, and thus development of a still more highly efficient kokumisubstances is possible.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. All the cited referencesherein are incorporated as a part of this application by reference.

The invention claimed is:
 1. A method for preparing a food or beveragethat contains a kokumi-imparting substance, the method comprising: A) afirst step of detecting the calcium receptor activating ability of atest substance, which is an indication of the ability to impart kokumi,by using recombinant cells which have been transformed with a foreigncalcium receptor gene, wherein the test substance enhances a tasteselected from the group consisting of salty taste, umami taste, sweettaste, sour taste, and combinations thereof; B) a second step ofmeasuring the kokumi-imparting effect of the test substance for whichcalcium receptor activating ability has been detected in the first step;C) selecting a substance which is effective to both activate the calciumreceptor and impart kokumi, and D) combining the substance selected instep C) with a food/beverage, thereby producing said food or beveragethat contains a kokumi-imparting substance.
 2. The method of claim 1,wherein the kokumi-imparting effect of the test substance is measured bya quantitative sensory evaluation test.